Evaluation of antioxidant and antibacterial properties of dehydrocostus lactone isolated from Echinops kebericho root

Abstract Background and Aim Echinops kebericho, an endemic plant to Ethiopia, traditionally used to treat infectious as well as noninfectious diseases. The primary objective of this study was isolating dehydrocostus lactone (DHCL) from E. kebericho and evaluating antibacterial activities on selected human pathogenic bacteria. Methods Extraction method used in this study was maceration. Based on the bioassay information methanol extract of the root of E. kebericho was subjected to column chromatography on silica gel by increasing solvent gradients to isolate DHCL. Optimized amount isolation of DHCL was done by dissolving methanol crude extract by hexane followed by recrystallization at room temperature in the dark place. Different concentrations of the extract were subjected by disc diffusion method against tested bacterial species and antioxidant activity test. Results The phytochemical analysis of E. kebericho revealed a high presence of terpenoids, which are diverse natural compounds known for their antimicrobial and antioxidant properties. This suggests that terpenoids contribute significantly to the pharmacological effects of E. kebericho. In antibacterial testing, Escherichia coli was the most sensitive bacterium among all extracts and concentrations. The methanol extract displayed higher antioxidant activity compared to ethyl acetate and hexane extracts, indicating a higher concentration of antioxidant compounds. Notably, the isolated compound DHCL showed promising activity against tested pathogens and significant antioxidant activity. The higher activity of DHCL compared to the crude extracts suggests its responsibility for the observed effects, indicating that the isolation and purification process may have concentrated its beneficial properties. These findings highlight the potential of E. kebericho and DHCL as sources of bioactive compounds for therapeutic applications. Conclusion All tested extracts and pure compound showed higher inhibition than positive controls in both bioassay. DHCL the principal bioactive component in the root extract of the plant and it displayed potent antibacterial and antioxidant activity.


| INTRODUCTION
Recently, diseases arising from emerging pathogens and sources of free radical initiators become a major treat for medical communities in public health. 1,2The spread of antibiotic-resistant bacteria worldwide becomes a substantial threat to morbidity and mortality. 3Mitigation of the current development represents one of the most significant challenges to modern medicine that increased the difficulty of treatment. 1,4In this concern, it could be responsible for more than 10 million human deaths per year worldwide by 2050. 5That forcing the use of drugs that are more noxious, costlier, and with low efficiency. 6ee radicals and oxidants are now a days becomes a global challenge due to the development of chronic and degenerative disease such as cancer, cardiovascular, autoimmune disorders, rheumatoid arthritis, and neurodegenerative diseases. 7The counteract mechanism of these oxidative stress can be either naturally generated in situ (endogenous antioxidants), or externally supplied through foods (exogenous antioxidants). 8The roles of natural metabolites (antioxidants) are to neutralize the excess of free radicals, to protect the cells against their toxic effects and to contribute to disease prevention. 9r primary healthcare more than 80% of people worldwide uses herbal medicine as a means of solution to treat diseases regarding microbial 10 and accepted as a source for discovering new potent phytochemicals that have been used to treat serious diseases. 11wadays, the urgent issue in the scientific community is designing a new approaches to deal with drug-resistant pathogenic bacteria. 12reover, researchers have been attracting to develop effective of antimicrobial agents by modification of the structure of antibacterial substances. 13,146][17] In this scenario, researchers are shifting to new alternatives to fight back this concerning situation. 6In developing countries preparation of herbal formulations for tackling diseases arising from bacteria as well as oxidants are a long history. 18hinops kebericho, endemic to Ethiopia, belongs to a family of Asteraceae. 19Traditionally, the smoke of root of this plant uses as the treatment of typhus and fever, moreover, stomach ache can be reduced by chewing the root of Echinops.Ethno-veterinary medicinal values of root of this plant have been reported as curing intestinal diseases in cattle.Smokes as snake repellent also pronounced in many parts of the country, Ethiopia. 20Antileishmanial 21 and antifungal 22 activities of this plant have been reported a potential candidate to treat these diseases and the presence of potentially active ingredients has been reported from the genus Echinops. 23,24e medicinal plant species considered in the present study were selected based on its high frequency of mention registered from different ethnobotanical surveys reported in the literature for the treatment of different diseases among endemic plants to Ethiopia.In addition, there is no enough reports of similar studies on pure isolated compound dehydrocostus lactone (DHCL).Therefore, the purpose of this study was isolating DHCL from root of E. kebericho to evaluating its antibacterial and antioxidant activities in comparison with different crude extracts.

| Plant material collection and extraction
The fresh plant material was bought from Segno Gebeya, Dessie City Administration, about 400 km away from Addis Ababa, during the month of September 2021.The producer reported by Deyno et al. 25 and Wondiafrash 26 were applied.The specimen was submitted to Addis Ababa University (AAU) National Herbarium of Ethiopia for identification and deposition voucher number.The sample was authenticated by Melaku Wondafrash and voucher number AAU-Herbarium-S1219 for E. kebericho was deposited at the National Herbarium of AAU, Ethiopia.
The appropriate research type (design) was comparative experimental design.The dried root (1 kg) was grounded in to coarse powder using electrical mill.One hundred and eighty grams of powdered sample was macerated with 1 L n-hexane, ethyl acetate and methanol for 72 h for each solvent with continuous shaking.The extracts were filtered through Whatman filter paper (No.1) and concentrated by Rotary evaporator at reduced pressure at 40°C.The resulting semidried extract was then stored in refrigerator below 4°C until used for antimicrobial and antioxidant activities and the preliminary phytochemical analysis.

| Phytochemical screening
The preliminary phytochemical analysis of the extracts was carried out using standard procedures to identify the various constituents.

| Test for alkaloids
Three milliliters of the extract was added to 5 mL of Hager's reagent (saturated picric acid solution).A yellow precipitate produced immediately an indication of alkaloids. 27

| Test for flavonoids
One milliliter of the extract, a 5 mL of dilute ammonia added and 1 mL conc.H 2 SO 4 .A yellow color was produced, which indicated the presence of flavonoids. 28

| Test for saponins
Three drops of the extract mixed with 5 mL of water.After 15 min form foam indicated the presence of saponins. 28,29

| Test for tannin
Three drops of test solution with gelatin solution white precipitate formed indicate the presence tannin. 30

| Test for carbohydrate
Three drops of test solution with five drops of Benedict's reagent (alkaline solution containing cupric citrate complex) boiled it in waterbath at 70°C for 10 min and formed a reddish brown precipitate showed the presence of carbohydrate. 31

| Test for steroids
One milliliter of the extracts was dissolved in 10 mL of chloroform and equal volume of concentrated H 2 SO 4 was added by sides of the test tube.
The upper was layer turned to red and the acidic layer showed yellow color with green fluorescence that indicates the presence of steroids. 31

| Test for coumarins
Two milliliters of extract added 3 mL of 10% NaOH yellow color formed.This showed the presence of coumarins. 323.8 | Test for terpenoids 0.5 mg of the extract was dissolved in 2 mL of chloroform and the addition of 2 mL of Conc.H 2 SO 4 .Then reddish brown color was formed which indicates the presence of terpenoids.32 2.3.9 | Test for phenols Three drops of test solution added 5% ferric chloride.Then blue black color formed which indicates the presence of phenols.33

| Test for anthraquinones
Three drops of test solution added five drops of 2% hydrochloric acid.
Appearance of red color indicated the presence of anthraquinones. 29

| Isolation of DHCL from methanol extract
Ten grams methanol extract was mixed with silica gel by methanol to adsorb the crude extract for column chromatography.After drying using rotary evaporator brown jelly extract was obtained and then the column was packed with silica gel and hexane.After 1 h, the adsorbed sample was applied to the top of column then the column was eluted using different solvent systems with increasing gradient.By this similar step, many fractions were collected and the TLC of each fraction was checked using different n-hexane and ethyl acetate solvent ratio.Ten fractions were obtained by mixing fractions with similar spot (similar Rf) and concentrated.Fraction 7 and 9 were observed a promising fraction with white needles crystals at the bottom of the collecting vials.Figure 1  Finally, these crystals were carefully filtered by Whatman filter paper and a characteristic single violet spots was observed on TLC upon using UV lamp after spraying 1% vanillin sulphuric acid and heating for a few minutes.The resulting purified sample was submitted to spectroscopic analysis.
The amount of DHCL isolated from column chromatography was so small to continue biological assay.So, a large-scale isolation technique was developed using methanol as an extracting solvent.
One thousand grams of powdered root of E. kebericho was extracted using 3000 mL of methanol in 5000 mL Erlenmeyer flask for 30 min over hot plate with magnetic stirrer.The crude extract was concentrated using rotary evaporator at reduced pressure to avoid methanol.The resulting crude extract was subjected to solventsolvent extraction using n-hexane and finally the extract was stored at room temperature under dark condition.After 24-h needle shaped crystals were collected and weighed to give 50 g of pure DHCL.

| Test organisms
The bacteria species selected for this study were two gram-positive namely Staphylococcus aureus and Listeria monocytogenes and two gram-negative Escherichia coli and Klebsiella pneumonia.These microorganisms were obtained from Wollo University Biology Department Laboratory.

| Media preparation
Thirty eight milligrams of MHA powder was added into 1 L of distilled water in a flat-bottomed conical flask.The media was completely dissolved by heating the mixture with frequent agitation until the clear solution was observed.The flask was covered with aluminum foil after tightly closed using cotton wool.
The mixture was autoclaved for 15 min at 121°C after which it was left to cool down to room temperature.Seventy milliliters freshly prepared sterile MHA medium was poured into 150 mm diameter Petri dishes.The Petri dishes containing the media were then placed in a sterile and properly adjusted refrigerator until bacteria inoculums were spread on them. 34 35To measure the turbidity of bacterial suspension spectrophotometer was used.Our absorbance of the suspension measurement was done at the wavelength to 600 nm.

| Determination of inhibition zone
The negative control (5% DMSO in water) and positive control (chloramphenicol, 100 μg/mL) were placed into the labeled agar wells.The plates were placed at room temperature for 2 h and then incubated at 37°C for 24 h.All tests were performed in triplicate for each bacterial species.Finally, the diameters of inhibition zones were measured in millimeter using digital meter.The mean zone of inhibition and standard error of the mean (mean ± SEM) were calculated for the fractions and controls.The zone of inhibition was F I G U R E 1 Extraction and isolation method.
analyzed whether the bacteria is resistant or sensitive to the reference of a particular antibiotic and values. 36

| Determination of antioxidant activity
Antioxidant activity of different leaves extracts of E. kebericho and DHCL were measured by 1, 1-diphenyl-2-picryl hydrazyl (DPPH) following the producer used by Hussen and Endalew. 27Briefly, 0.1 mM solution of DPPH was prepared by dissolving 0.004 g of DPPH crystalline solid in 100 mL of analytical grade methanol and stored at 4°C.A 3 mg of the plant extract was dissolved in 10 mL of methanol to prepare 300 μg/mL stock solutions and then serial dilution with methanol was performed to prepare the required concentrated solutions (50, 100, 150, 200, 250, 300 μg/mL) followed the protocol proposed by Hussen and Endalew. 27A 2 mL of plant extract solution from each concentration was taken in a test

| Statistical analysis
All tests were carried out in triplicate, and data was expressed as mean standard deviation (SD) or standard error of mean (SEM), excel, and Chemdraw (to draw different chemical structure).
Differences among means at 5% level (p < 0.05) were considered statistically significant.

| Percentage yields and phytochemical screening tests of extracts
The yield of the crude extracts obtained from root of E. kebericho  various secondary metabolites, such as alkaloids, flavonoids, coumarins, phenols, terpenoids, tannins, and steroids, but there was negative result on carbohydrate and saponins (Table 1).

| Characterization of DHCL
DHCL was isolated as white crystals (Rf 0.36 in EtOAc:Hex  All olefinic carbons were detected as exocyclic double bonds. The other characteristics signal that appeared at δ 85.34 ppm was an oxymethine carbon of the lactone ring.Thus, on the basis of spectroscopic data and also comparison with the literature data, 37 structure of the compound was deduced as dehydrocostus lactone (DHCL).

| Antibacterial activity
Antibacterial activity of crude extracts and purely isolated DHCL against selected bacteria was evaluated using disc diffusion methods (Table 3).The extracts and the isolated compound were tested against selected pathogens.Figure 4 showcases the antibacterial activity of the extracts and DHCL and that enables a comparison of the antibacterial efficacy between different extracts and DHCL, providing valuable information on their potential as antibacterial pneumonia and E. coli because of the nature of the cell-wall. 38It is believed that the inhibitory effects of bioactive compounds are dependent on its ability to damage cell membranes. 39Among the tested organisms, S. aureus was observed as highly susceptible in a plant extracts.
The bioassay-guided fractionation of the methanol extract and further purification of the most antibacterial active fraction led to the isolation and identification of an antibacterial sesquiterpenes lactone, DHCL.As shown in Table 3, this compound exhibited potent antimicrobial activity against tested bacterial species and showed the highest antibacterial activity than both extracts and positive control, chloramphenicol.The data obtained from the present study is comparable with reported data regarding the activity of crude extracts. 25The synergic effect of phytochemicals as shown in Table 2 makes all the extracts promising and potent antibacterial agent compared with the standard chloramphenicol.In this study, the pure isolated compound DHCL showed higher activity than all the extracts as well standard.

| Antioxidant activity
The antioxidant activity of root extracts and DHCL has been studied by its ability to reduce DPPH.Interaction of antioxidant compounds with DPPH is based on the transfer of hydrogen atom or electron to DPPH radical and converts it to 1, 1-diphenyl-2-picrylhydrazine. 27The result of reduction DPPH radicals proved by the transformation of purple color to yellow pale color, which demonstrates the scavenging activity. 40e antioxidant activity of the three solvents extracts, DHCL and ascorbic acid against DPPH assay was tested with concentrations ranging from 50 to 300 μg/mL as the results shown in Table 4.As shown in illustrates the extraction and isolation method used in the study.The diagram provides a step-bystep visual representation of the process, highlighting the key stages and techniques involved in obtaining the desired compound, DHCL.
Crude extracts (3.5 g) were dissolved in DMSO until the volume of the solution became 1 mL to get 3.5 g/mL stock solutions.Different concentrations of extracts with different solvents (200, 100, 50 mg/mL) were prepared after dilution of the stock solution with DMSO.Fresh culture bacteria were suspended into 5 mL of sterile normal saline water and then turbidity of suspension was adjusted equivalent to 0.5 McFarland standards by reading on McFarland Densitometer instrument.A sterile cotton swab was dipped into bacterial suspension, rotated gently and pressed firmly on the inside wall of the tube above the fluid level to remove excess inoculums from the cotton swab.The swab was streaked to the entire surface of the MHA plate three times by rotating approximately 60°C each time to ensure an even distribution of the inoculums.Petri-plates were left for 3 min at room temperature.Sterilized filter papers disk containing different concentrations of plant extract were placed on plate containing MHA.
(1:1); mp 56-57oC) with a molecular formula C 15 H 18 O 2 as evidenced by LC-ESI-MS which exhibited a molecular ion peak, [M] + , at m/z 230.96(100%).The IR spectrum showed characteristic absorptions attributable to conjugated ketone group (1744 cm −1 ) and C = C (1644 cm −1 ). Figure 2 presents the ¹H NMR spectra of DHCL.The peaks in the spectra correspond to different hydrogen atoms in the DHCL molecule, providing valuable information about the molecular structure and chemical environment.

The 1 H
NMR spectrum (Table2) displayed the six exocyclic olefinic protons of which four are observed as doublet because of geminal coupling and were appeared as single.A triplet signals at δ 3.94 (1H, t, J = 9.2) was assigned for proton attached on oxymethine carbon.The remaining multiplet signals at different chemical shifts were assigned for methylene protons on C2, C3, C8, and C9 and Methine protons C1, C5, and C7.Figure3displays the ¹³C NMR spectra of DHCL.The peaks in the spectra correspond to different carbon atoms in the DHCL molecule, providing insights into the molecular structure and composition.The 13 C NMR spectrum (Table 2) of an isolated compound displayed 15 distinct peaks accounted for by 4 methine, 7 methylene and 4 quaternary carbons in the 135 DEPT spectrum.The most downfield signals at δ 170.31 was a characteristics peak of α-β-unsaturated lactone functional group.Six olefinic carbons were recorded at a chemical shift of δ 151.35, 149.25, 139.71, 120.27, 112.60, and 109.54 which were assigned for C-4, C-10, C-11, C-13, C-14, and C-15, respectively.

Figure 5 demonstrates
figure allows for a comparison of the antioxidant capacity between different extracts and DHCL, offering insights into their potential as antioxidants.

F I G U R E 5
Antioxidant activity of extracts and dehydrocostus lactone (DHCL).Evaluation of antioxidant and antibacterial properties of DHCL Isolated from Echinops kebericho Root.

1
Phytochemical screening tests of n-hexane, ethyl acetate and methanol extracts.
F I G U R E 2 1 H NMR spectra of dehydrocostus lactone.ENDALEW ET AL.|5 of 11tube and then, 3 mL of DPPH solution was added in each test tube.The same procedure and concentrations were prepared for isolated pure compound, DHCL.After 30 min incubation in the dark, the absorbance at 517 nm was recorded using a UV-Vis Spectrophotometer.where,A control is the mixture of methanol/ethanol/water and DPPH solution, and A sample is the mixture of sample extract and DPPH solution.
13C NMR data for DHCL and their literature.
methanol, ethyl acetate and n-hexane were 19%, 12% and 9%, respectively.The methanol crude extract had the highest percentage yields compared to ethyl acetate and n-hexane crude extracts.The result of percent yield shows the test plant contains more polar constituents than nonpolar ones.As the phytochemical analysis indicated extracts of E. kebericho is rich inT A B L E 2

Table 4
, both the EtOAc, MeOH and DHCL were displayed a comparable and significant concentration-dependent free radical scavenging activity from 91.05% to 92.84%, 91.81% to 93.06%, and 93.45% to 94.48%, respectively, compared with that of T A B L E 3 Comparison of antibacterial activities (zone inhibition) of crude extracts and isolated compound (DHCL) (mean ± SEM mm diameter).Antibacterial activity of extracts and dehydrocostus lactone.0.812 65.13 109.70 0.165 91.05 95.34 0.127